Regulation of p42/44 mitogen-activated protein kinase by G-protein coupled receptors

Burt, Andrew Robert (1997) Regulation of p42/44 mitogen-activated protein kinase by G-protein coupled receptors. PhD thesis, University of Glasgow.

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The cascade linking growth factor receptors and G-protein coupled receptors (GPCRs) to the regulation of p42/44 mitogen-activated protein kinase (p42/44MAPK) has been described in some detail and involves numerous steps of phosphorylation and protein- protein interaction. There is a remarkable degree of similarity in the pathways initiated by these two receptor families, although some degree of cell/receptor specificity exists. In this study, the regulation of p42/44MAPK by the alpha2A adrenoceptor and the delta opioid receptor following transfection into Rati fibroblasts to different receptor densities was quantitatively measured and compared to the regulation by endogenous G-protein (lysophosphatidic acid: LPA) and tyrosine kinase (epidermal growth factor: EGF) coupled receptors. The wild type alpha2A adrenoceptor was expressed at a high (clone TAGWT 17: 5600 fmol/mg protein) and low density (clone TAGWT 3: 460 fmol/mg protein) which permitted greater G-protein activation in clone TAGWT 17 cells as measured by agonist (UK14304)-mediated stimulation of high affinity GTPase activity and cholera toxin- catalysed [32p]ADP-ribosylation of Gi-like proteins. Greater maximal activity and lower half-maximal agonist concentrations (EC50 values) were determined for clone TAGWT 17. The greater signalling ability was also determined at the level of inhibition of adenylyl cyclase. Previous studies had demonstrated the activation of p42/44MAPK by the alpha2A adrenoceptor. UK14304 treatment of cells of both these clones phosphorylated a significant proportion of p44MAPK, demonstrating that there was no requirement for high level expression to observe this coupling. This response was dose-dependent with TAGWT 3 clone displaying a 10-fold greater EC50 value than clone TAGWT 17. An Asn79 mutation of the alpha2A adrenoceptor (Asp79 changed to Asn) substantially reduced the G-protein coupling efficiency of the receptor, previously demonstrated as a complete or partial loss of signalling capacity. This mutant receptor expressed in fibroblasts (clone ASN79 4; 3000 fmol/mg protein; ASN79 9: 4300 fmol/mg protein) demonstrated an attenuated, but not eliminated, ability to stimulate G-protein activation, inhibit adenylyl cyclase and phosphorylate p42/44MAPK. This indicated that the coupling efficiency to the MAPK cascade was not exceptionally poor, as even a severely limited G-protein activation substantially stimulated this pathway. However, the time course of phosphorylation of p44MAPK was significantly more delayed and transient which would indicate that the kinetics of this regulation is dependent on the level of stimulation of the G-protein-coupled pathway. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Milligan, Professor Graeme
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-71325
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 25 Aug 2022 09:02
Thesis DOI: 10.5525/gla.thesis.71325

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